The Methods Employed in Mass Spectrometric Analysis of Posttranslational Modifications (PTMs) and Protein–Protein Interactions (PPIs)

Abstract

Mass Spectrometry (MS) has revolutionized the way we study biomolecules, especially proteins, their interactions and posttranslational modifications (PTM). As such MS has established itself as the leading tool for the analysis of PTMs mainly because this approach is highly sensitive, amenable to high throughput and is capable of assigning PTMs to specific sites in the amino acid sequence of proteins and peptides. Along with the advances in MS methodology there have been improvements in biochemical, genetic and cell biological approaches to mapping the interactome which are discussed with consideration for both the practical and technical considerations of these techniques. The interactome of a species is generally understood to represent the sum of all potential protein-protein interactions. There are still a number of barriers to the elucidation of the human interactome or any other species as physical contact between protein pairs that occur by selective molecular docking in a particular spatiotemporal biological context are not easily captured and measured.

Current Methods of Post-Translational Modification Analysis and Their Applications in Blood Cancers

“…Other techniques often used to study the proteome include flow cytometry, protein microarrays, Western blotting, and enzyme-linked immunosorbent assays (ELISA) ( Figure 2 ) [ 1 ]. Additional experimental steps are often required to optimize proteomic techniques for PTM analysis due to the substoichiometric, transient and labile nature of PTMs which hinders the maintenance and detection of modifications during analysis [ 2 ]. To overcome the issue of low stoichiometry, enrichment of the PTM of interest is often performed prior to MS analysis.…”

“…Ubiquitination plays a key role in many cellular processes such as signal transduction, transcriptional regulation, DNA repair, intracellular localization and, most notably, the tagging of proteins for transfer to the proteasome for degradation. Abnormal ubiquitination can lead to enhanced or reduced degradation of specific proteins, hyperactive or inactive cell signaling pathways and an overall altered homeostasis in the cell [ 2 ]. A multienzyme cascade constituting ubiquitin-activating (E1), ubiquitin-conjugating (E2) and ubiquitin-ligating (E3) enzymes, results in the covalent attachment of the 76 amino acid protein, ubiquitin (Ub), to a lysine residue on the target protein.…”

“…There are various types of glycosylation, the most common being N-glycosylation followed by O-glycosylation. This PTM is one of the most complex PTMs due to the heterogeneity of glycan structures and their glycosylation sites [ 2 , 48 ].…”

Peptide array–based interactomics

“…As peptides are chemically synthesized, any PTM can be included as long as it is compatible with the synthesis technique. The binding surface is thus fully modified, which is usually not the case with complete proteins [ 33 – 36 ].…”

“…Fast regulation of biological networks relies on the rapid addition and removal of PTMs during signaling, leading in many cases to the formation or loss of protein interactions. Capturing these transient interactions is challenging [ 36 , 37 ]. Dissecting the recruitment of proteins using PTM-containing peptides allows identifying different complexes involved in the interaction (Fig.…”